A multi-proxy bioarchaeological approach reveals new trends in Bronze Age diet in Italy

This study investigates changes in dietary practices and subsistence strategies in Bronze Age Italy integrating isotopic analyses with archaeobotanical and archaeozoological data. By investigating food habits, we contribute to reconstructing human lifestyles and highlighting possible links with the economic/social organization when the rise of stratified societies and new economic activities affected subsistence practices. Stable isotopes analyses in humans and animals were performed on 6 Italian sites dating from 2300 to 900 cal. BC, followed by a complete review of additional 19 sites, which forms the basis of a diachronic and geographic comparison for Bronze Age Italy. The geographic analysis shows a more varied diet in northern and central Italy, compared to the south. The diachronic analysis highlights the homogeneity of food habits during the Early Bronze Age, contrary to the later phases when an increase in dietary variability and a higher animal protein consumption are revealed. The Middle Bronze Age appears as a pivotal moment in protohistoric societies, a phase of transition. The consumption of different foodstuffs highlights the importance of cultural exchanges, resulting in a sort of “food globalization”, although environmental and climatic fluctuations could also have affected dietary patterns, favoring some crops over others.

www.nature.com/scientificreports/ In central Italy (Toscany and Lazio), husbandry was more focused on cattle, sheep and goats and less on pigs, e.g. Petrosa 44,45 . In this area at the beginning of the MBA, the animal economy was focused on the breeding of cattle, however from the late MBA husbandry strategies changed and caprines became predominant. This trend continued throughout the RBA and FBA up to the early Iron Age, when pigs become more significant 46 . During the last phases of the Bronze Age, pastoral activities seem to increase and caprines become the main species at the sites of Marche and Abruzzo 47 . Research in the Marche region during the MBA suggests a mixed type economy. Nevertheless, some settlements may have had a more pastoral economy, while others had subsistence strategies based on agricultural practices associated with cattle and pig husbandry 48 . For this region too, it seems that there was a marked increase in caprines mainly used for meat. Moreover, it seems that the surplus of sheep could be interpreted as a prestigious goods of the emerging elites 43 .
In southern Italy, studies on sites such as Broglio di Trebisacce 49 , Torre del Mordillo 50 and Coppa Nevigata 51 allow a diachronic analysis of of changes in the animal economy. However, the lack of information for the EBA constitutes a substantial gap 52 . Higher percentages of caprines compared to cattle and pigs have also been observed in southern Italy for the MBA and RBA where sheep were predominant 43,52 . Even though they were mainly exploited for meat, secondary products were important for the local economy, as recorded in Puglia and Sibaritide 49,50 . Notwithstanding, the sites with high percentages of cattle are numerous, e.g. Torre dei Passeri 53 , Monopoli 54 , and Bari S. Maria del Buon Consiglio 55 . These animals were no longer being reared exclusively for meat and other species such as sheep or pig also provided good quality meat. Occasionally game, i.e. deer, may also have purposefully supplemented meat production 37 . The increase of the number of calves in the animal records suggests an evolution of the dairy industry and the general prosperity of settlements 43 . In some of the southern sites, hunting was still significant for the local economy 54,56 and seems to have intensified at the end of the Bronze Age. This phenomenon could be linked to increased demands for meat following a rise in the exploitation of secondary products 43 .
Evidence for aquatic species is rare for the Bronze Age sites of the Peninsula. When present, mainly freshwater and euryaline species are found in the north and in central Italy 57 . Pike and turtle have been found in the Lagazzi pile-dwellings and fishing is attested by tench, pike and rudd remains at Canàr 57,58 . Dating to the FBA and early IA, abundant remains of tench and pike have been found in Frattesina 57 . In the South of the Peninsula, the aquatic remains are mainly of saltwater species 56 and the main discoveries are from the Apulia coast (Monopoli, Roca, Punta Le Terrare) 56 .

The archaeological setting
The cemeteries of Pertuso, Buco del Diavolo, Ostiglia La Vallona, Gr. Vittorio Vecchi, Trinitapoli and Castiglione presented here for the first time are shown in Fig. 1 and Table 1. The selection of the sites was driven by the aim at increasing the representability of each chronological period and geographical area. It takes into account the limitations due to lesser explored areas, i.e. the center and south of the Peninsula, and the changes observed in funerary practices with the passage from inhumation to cremation during the MBA/FBA, which curb the analysis of bone collagen. The paucity and heterogeneity of the information provided by some of the newly discovered sites restricts this study, nevertheless they contribute substantially to the reconstruction of a dietary overview.
The sites of Pertuso, Buco del Diavolo, Ostiglia La Vallona, Gr. Vittorio Vecchi, Trinitapoli and Castiglione, located in the north, center and south of the peninsula, span from EBA to the FBA/IA. These cemeteries include 164 humans and 51 domestic and wild animals recovered in association with the human remains, then they are all coeval. Each archaeological site context is detailed in Supplementary Text A.1.

Results
The carbon and nitrogen isotopic ratios from Pertuso, Buco del Diavolo, Ostiglia La Vallona, Gr. Vittorio Vecchi, Trinitapoli and Castiglione are presented in Supplementary To minimize the environmental differences, the humans have been compared considering the offset between the median of the humans and that of the associated domestic animals for each site to evaluate differences in the diets of human communities (Δ 13 C h-f and Δ 15 N h-f ). Indeed, considering that the diet-tissue trophic enrichment factor is estimated to be around 3-5‰ for the nitrogen and 0-1‰ for the carbon per trophic level 66,67 , the offsets between the human and fauna are good indicators of a community diet (protein intake).
The sites in Liguria, Pertuso and Buco del Diavolo, are dated to the EBA and FBA/EIA respectively. The two animal groups present typical ranges for a temperate environment predominantly dominated by C 3 plants. When considering the herbivores of both sites, no significant differences occur for both δ 15 N and δ 13 C values (δ 15 N, p = 0.361; δ 13 C, p = 0.854). This suggests that the two sites were placed in a similar environment and that no great environmental and/or paleoclimatic changes occurred during the Bronze Age in this area and that herbivores consumed similar plants. Since the two animal assemblages show the same food pattern, the two human groups are directly comparable. If any difference in dietary patterns appears between the two groups, it will likely be the result of food preferences rather than of a "different environmental offer". Pertuso (Liguria). The δ 15 N human median is 7.2‰ (min = 6.5‰, max = 7.9 ‰, n = 14) and the δ 13 70 . Paleopathological analyses have also shown that adults present stress marks linked to their growth, possibly related to food deficiencies as well (dental hypoplasia, cribra cranii and orbitalia) 70 . All evidence seems to support that this community was exposed to repeated stress, especially during childhood. Similar cases in which the δ 15 N and δ 13 C results of juveniles are lower than for the rest of the population have been recorded in other sites in Liguria and in southeastern France for the Neolithic 71,72 . Studies conducted on western Ligurian attest that Bronze Age communities were composed of small family groups 73 . Osteological studies on Pertuso humans seem to confirm this hypothesis, as olecranon perforations have been recorded in a number of individuals, supporting a certain degree of genetic links. The main settlements were in all likelihood scattered in naturally defended positions or consisted of fortified sites which controlled the surrounding area, e.g., Castellaro di Uscio 74 . As such, the archaeological evidence indicates an economy based on livestock and on the available local resources, as highlighted by the hunting, i.e. deer and wild boar, and gathering, i.e., apples, horns, acorns, that certainly enhances the evidence for a undifferentiated diet 73 . Figure 1. Location of the Italian Bronze Age cemeteries for which stable isotope analyses on humans are available (the sites from the literature are in black and the sites analysed here are in red). The map is elaborated using "Natural Earth. Free vector and raster map data @naturalearthdata.com" available at https:// www. natur alear thdata. com/ downl oads/ 10m-raster-data). Taking into account the local domestic animals, humans have an enrichment of 3.7‰ for nitrogen and 0.8‰ for carbon. These offsets indicate a varied mixed terrestrial diet, which includes both plant-mainly C 3 , and animal resources, with the exception of BD1092, whose δ 13 C value (− 18.5‰) suggests a mixed C 3 -C 4 plants consumption. It is likely that meat and dairy products originate principally from sheep and goat, the most represented at the site 65 . Nevertheless, the contribution of other domestic species, like cattle and pigs, should not be underesti- www.nature.com/scientificreports/ mated. The similar isotopic results of these species and caprines makes it difficult to identify which species were predominantly consumed by the humans. The second group includes BD876, BD1132, BD882 and BD794. The median values are 8.7‰ for δ 15 N (min = 8.5‰, max = 9.1‰, n = 4) and − 15.6‰ (min = − 16.5‰, max = − 15.0 ‰, n = 4) for δ 13 C. For both isotopes, significant offsets are evident between the humans and the domesticated animals (∆ 15 N h-f = 5.2‰ and ∆ 13 C h-f = 5.0‰). The relatively high δ 15 N values suggest a substantial animal protein intake and aquatic foodstuffs could have been part of the diet even though fish remains have not been found in the site. The δ 13 C values, enriched in 13 C, support an important C 4 plant consumption. Considering the carbon and nitrogen ratios, marine foodstuffs could have been part of the diet as well, but the altitude and the geographical position of the site make this option quite improbable. This evidence confirms that millets played an essential role in the diet, showing important differences in food choices between the two groups. The first archaeobotanical evidence for millets in Liguria comes from the MBA (Bric Tana) 75 , however the most consistent finds are dated to the FBA-early IA (Monte Trabocchetto) 17 . Consequently, considering the radiocarbon dates of the second group 65 , the δ 13 C values enriched in 13 C support an important consumption of millets from the FBA-IA transition, consistent with the dates of the archeobotanical results.
The presence of two groups with different diets at Buco del Diavolo is not dependent on sex or age of the individuals because both groups consist of men and women of different age. Therefore, the first explanation to justify this variability could be related to social reasons. Food choices can be a sign of individuals' differential access to resources, making these choices indirect evidence for the stratification of the community. Lightfoot et al. 76 highlighted the consumption of millets for some individuals during the Bronze Age (Nadin-Gradina, Croatia) and proposed millet as a low status foodstuff after considering the relationship between δ 13 C and burial type. At Buco del Diavolo, some bracelets and a bronze torque are indicative of people of high social status. Unfortunately, as skeletal remains were not articulated, it was not possible to associate the grave goods to the individuals, but it is possible that such differences may also be reflected in diet.
The second explanation could be related to cultural reasons. A viable alternative is that the two groups had different origins and in accordance different dietary habits. Millet was already consumed since the MBA in northern Italy (Verona area) 59 . Therefore, it is likely that some individuals from this area had moved to Liguria. Some archaeological elements typical of the southwestern alpine region are present in the Po basin, in the Provencal areas, in Switzerland, Germany and in southeastern France (RSFO Group of the Urnfield Culture), as attested by bronze grave goods 77 . Thus, the growing exchanges led to an increasing mobility, which could be reflected in different diets.
A third explanation could be associated to the different chronology. The first group is dated to the FBA, while the second group to the beginning of the IA 65 . It is therefore possible that the diets could be the result of different agricultural practices during the two periods: cultivation of C 3 plants during the FBA and more C 4 plants during the IA. Moreover, a rapid climate change between 1550-550 cal. BC across the Northern Hemisphere has been recorded 78 and the paleoclimatic fluctuations responsible for the increase in drier conditions could have led to the diffusion of new crops, like millets. In addition, the spread of human activity affected the ecosystems leading to a decline in woodland cover, the expansion of shrubland and grassland and an increase in soil erosion. There is also evidence for burning from the Late Bronze Age onwards 79 . At present, none of these hypotheses can be excluded, further research on additional isotopes, such as Sr to study the mobility, should provide a clearer picture.
Ostiglia La Vallona (Lombardia). The δ 15 N human median is 10.7‰ (min = 10.1‰, max = 13.3‰, n = 5) and the δ 13 C median is − 13.0‰ (min = − 13.3‰, max = − 12.6‰, n = 5). The offsets between humans and animals are 4.4‰ for nitrogen and 8.0‰ for carbon. These results indicate an important consumption of enriched 15 N and 13 C resources. The high carbon ratios suggest C 4 plants consumption like millets, as confirmed by coeval and nearby communities (Olmo di Nogara, Bovolone) 25 , whereas the enrichment in 15 N could highlight an important animal proteins intake as well as aquatic foodstuffs. Indeed, local communities have exploited the aquatic environment since the EBA-MBA transition, as confirmed by the freshwater animals recovered in the pile-dwelling and Terramare settlements 57 . The isotopic values of freshwater fish from Fondo Paviani 25 are further evidence of the possible contribution of these resources to the human diet.
Moreover, plants enriched in 15 N, like the modern cattail (Typha latifolia), a species typical of marshy environments (δ 15 N x = 7.0‰, n = 79) 80 could have been part of the diet, and its consumption is attested since the Mesolithic 81 . Off-site palynological studies in the lower Veneto plain and in Lombardy show a higher rate of afforestation (30-50%), with extensive presence of wooded or marshy areas in the Bronze Age [81][82][83][84] . Consequently, wetlands with their typical fauna and vegetation, like bulrush 12 , were potentially exploited for food resources.
According to the archaeological fish isotopic data from the Mediterranean 68,69 , marine resources could have been consumed as well. If marine foodstuffs were consumed, this could lead to the enrichment of both human δ 15 N and δ 13 C values, consequently limiting the importance of C 4 plants in the diet. However, when considering the local archeozoological evidence and the geographical position of the site, a freshwater food intake is more likely alongside an important consumption of C 4 plants.
Notwithstanding the limited number of individuals, no dietary differences were detected according to age groups. The only exception is OLV371sub, who shows an enrichment in 15 N compared to rest of the group but the young age of this individual (1-1.5 years old) suggests that this could be associated to breastfeeding. The only individual whose sex has been estimated is OLV183, determined as male. This individual has the lowest δ 13 C and δ 15 N values. Given the lack of sex data for the other individuals, it is not possible to compare diet between the two sexes. www.nature.com/scientificreports/ Grotta Vittorio Vecchi (Lazio). The δ 15 N human median is 6.4‰ (min = 5.7‰, max = 6.7‰, n = 4), and the δ 13 C median is − 19.6‰ (min = − 20.1‰, max = − 19.2‰, n = 4). Concerning the ∆ 15 N h-f , an enrichment of 1.8‰ is recorded. This value is lower than expected, suggesting that the animal protein intake was rather limited. In addition to this, even though the assemblage is based on a low number of records, it seems that legume consumption was likely significant. This hypothesis would be supported by the botanical findings. Vicia faba seeds represent 57.3% of the total seeds (2182/3811) 85 . Notwithstanding, it is difficult to assess whether the legume intake was direct or indirect, this because some herbivores show low nitrogen values too and they were likely fed with legumes. Concerning the ∆ 13 C h-f , an enrichment of 0.9‰ is recorded. This result suggests that animal proteins were part of the diet. However, the small offset recorded for nitrogen and the likely important legume intake highlight that meat and dairy products were probably not a significant component of the diet.
The carbon and nitrogen data indicate that the individuals we analysed show a homogeneous terrestrial diet as the δ 13 C and δ 15 N are within a narrow range. The plant component was significant and mainly represented by cereals and legumes, as archaeobotanical studies confirm. Based on the available data, the δ 13 C and δ 15 N values do not seem to support the consumption of aquatic resources. Further isotopic investigations conducted on coeval individuals of this area will be necessary to confirm this main dietary pattern where a low animal protein intake and a likely important legume consumption is highlighted. Interesting, some new data from the area seem to confirm it 86 .
A comparison of the adults with the only adolescent shows the latter had the lowest nitrogen value (5.7‰) and the maximum carbon value (− 19.2 ‰). It is likely that this individual had a smaller animal protein intake than the others, but since the contribution of legumes to this group's diet is important, the picture could be much more complex.
Trinitapoli (Apulia). The data obtained by the present study are considered together with the seven individuals analysed by Arena et al. 63 . The δ 15 N human median is 8.3‰ and δ 13 C median is − 19.5‰. The δ 15 N results range between 6.5‰ and 10‰ (n = 14) and δ 13 C values between − 20.0‰ and − 18.7‰ (n = 14). Considering the human and animal offsets, ∆ 13 C h-f is 1.0‰ and ∆ 15 N h-f is 1.6‰. The carbon enrichment fits into the range between two consequent trophic levels, while the nitrogen one is lower than expected. These results suggest that, even if terrestrial foodstuffs from a C 3 environment were mainly consumed, animal proteins were not a major source, indicating a diet mainly based on plant proteins, including legumes, as attested by the archaeobotanical records of the area 7 .
Despite the consistency of the results, TRIAB20 and TRIAB22, differ from the main group, showing lower δ 15 N values and highlighting a lower animal protein intake. Tafuri et al. 59 identified a male with similarly low values at Toppo Daguzzo, suggesting the consumption of similar resources. Several hypotheses can explain these data: (I) a possible common origin for the individuals, explaining analogous dietary patterns; (II) a similar social status-the grave goods associated with the Toppo Daguzzo human indicate this individual was probably a warrior as he was buried with weapons 59 . Regarding the two women of Trinitapoli, it is difficult to associate the grave goods with the individuals because of the collective burials, thus it not possible to define social status except in specific cases 87 . Further investigations to reconstruct their origins could provide additional elements helping to explain these dietary differences.
No significant differences occur between the dietary patterns of the humans from the two sectors of the necropolis, AB and C (δ 15 N, p = 0.805; δ 13 C, p = 0.736). Similarly, no significant differences occur between individuals of different sex (δ 15 N, p = 0.28; δ 13 C, p = 0.97).
Castiglione (Sicily). The δ 15 N human median is 7.5‰ (min = 6.3‰, max = 9.3‰, n = 61) and the δ 13 C one is − 19.2‰ (min = − 19.7‰, max = − 18.9‰, n = 61). These values are indicative of a terrestrial diet and, even though no fauna is available for this site nor for the whole island for Later Prehistory, results show that most of the protein intake comes from plants from a C 3 environment. Nevertheless, humans show a large δ 15 N range suggesting a diversified intake of animal protein. As already highlighted for Trinitapoli, the individuals with the lower nitrogen values could indicate they had a significant legume consumption. The importance of agricultural foodstuffs is supported by studies on caries and other oral observations on the teeth of these individuals 88 . Statistical tests between individuals of different burials show no significant differences for δ 13 C (p = 0.355, H = 6.683) but differences emerged for δ 15 N (p = 0.003, H = 17.756). Significant differences have been detected between the following burials: TB96-TB93 (p = 0.035), TB95-TB93 (p = 0.046), TB119-TB96 (p = 0.041) and TB119-TB95 (p = 0.028) (p-values corrected by FDR). It is likely that the tombs reflect social organization, highlighted by a differential access to resources. For instance, the presence of a bossed bone plaque (ossi a globuli), interpreted as a prestigious object, in TB93, could imply that the individuals in this burial were of higher social status than those in the other tombs. Another hypothesis suggests a different origin for some of the individuals. Archaeological data support contacts between Sicily and the Aegean area 89,90 . Moreover, the general heterogeneity of the metric traits and, at the same time, similarities of some of these traits between Castiglione and groups from Greece and other Mediterranean countries, support migration events 91 , as also highlighted by other of southern Italy sites 92 .
Sex and age were estimated only for some individuals. Nevertheless, these parameters do not seem to have influenced food choice. Due to the small number of assessments, statistical tests are only possible for age categories (adults vs. juveniles) and no significant differences occur (δ 15 N p = 0.569; δ 13 C p = 0.56), advising that age did not affect food habits.  The Early Bronze Age. The Early Bronze Age sites, Arano, Ballabio, Dossetto, Mereto, Pertuso, Sedegliano, Gr. Scoglietto and Castiglione, have provided a total of 165 human and 47 animal samples, animal data being provided by six sites (Fig. 2).
Before comparing the humans, the domestic and wild herbivores and omnivores of each site were statistically compared. Significant differences occur among these groups (Supplementary Table A.5), likely linked to the geography and the landscape where the cemeteries are situated. Nevertheless, the animals mainly present a C 3 terrestrial diet with only small variations, since most of the animal ranges of the sites overlap. The nitrogen values span from 2.8 to 6.1‰ (excluding the fox from Gr. Scoglietto), indicative of the consumption of mostly unmanured resources 93 . The δ 13 C values are widespread, but the 89% of them (41/46) are included in a rather narrow range, spanning from − 22 to − 19.2‰. The five outliers, four cattle (two from Arano, one from Mereto and one from Gr. Scoglietto) and one deer (from Arano) have high δ 13 C values, indicating a C 4 plant contribution. Concerning Gr. Scoglietto, this evidence is in line with human dental calculus studies, where the presence of millet starch grains and phytoliths have been recorded 94 . The recovered phytoliths may be attributed to different cultivated crops like millets, but also to barnyard grass. In the latter case, grazers like cattle could have eaten C 4 plants in an anthropogenic environment. For Arano, the fact that a wild animal shows values enriched in 13 C suggests that C 4 non-cultivated plants where present in the local environment and wild animals could easily access them.
To minimize the environmental variability, we have compared the offset between the human and domestic animal medians to evaluate differences in the food choices (Supplementary Table A.8) and some patterns are evident. Excluding Dossetto and Sedegliano for δ 13 C values and Gr. Scoglietto for δ 15 N values for which greater offsets are recorded, most of the human groups show a homogeneous mixed terrestrial diet based on animal and C 3 plant proteins. In fact, the Δ 15 N h-f spans from 2.9 to 3.7‰ and the Δ 13 C h-f from 0.0 and 0.7‰, ranges that fit within the known offsets between two consecutive trophic levels.
A more detailed analysis reveals a uniform distribution of the δ 15 N data for Pertuso, Ballabio, Arano, Mereto, Castiglione and Sedegliano. All the sites show a quite wide range, spanning from 6.3 and 10‰, likely due to a diverse meat and/or dairy products consumption within the communities. The only site which significantly differs from the others is Gr. Scoglietto, where aquatic resources intake has already been suggested 26 . The location www.nature.com/scientificreports/ of Gr. Scoglietto in a coastal zone and in a lagoon-like area may have led the population to exploit the aquatic environment at the expense of agricultural products. Furthermore, the dental calculus results support the absence of any leguminous species 94 . The apparent absence of these plants in the diet associated to an important intake of animal foodstuffs can explain the enrichment in 15 N.
In terms of the carbon results, most sites (Arano, Pertuso, Ballabio, Mereto and Gr. Scoglietto) show δ 13 C values falling within a relatively narrow range, from − 21.0 to − 19.7‰. This distribution suggests a diet based on C 3 terrestrial resources, such as einkorn, emmer and bread wheat and barley associated to wild species differently available according to the regions (e.g., Quercus sp, Fucus carica, Juglans regia). Sedegliano, Dossetto and Castiglione show higher δ 13 C values compared to the previous ones. If for Castiglione the slightly higher δ 13 C values could be related to its southern geographical position and to a drier environment 95 , regarding Sedegliano and Dossetto, the higher enriched δ 13 C values are probably due to millets intake. These are likely the first isotopic evidence for millet consumption in Italy and this result is essential to trace the introduction of C 4 plants to the human diet.
The Middle Bronze Age. The Middle Bronze Age includes 13 sites: Olmo di Nogara, Bovolone, Fondo Paviani, Gr. Misa, Felcetone, Gr. Vittorio Vecchi, Gr. Regina Margherita, Lavello, Toppo Daguzzo, Trinitapoli, Gr. della Monaca, Gr. dell' Antenato and Murgia Timone, for a total of 157 humans and 61 animals. This period seems to be a phase of change according to the varied results for all the humans and animals (Fig. 3).
Both carbon and nitrogen animal group data are widespread and statistical differences occur (Supplementary Table A.5). The animals from northern Italy (Olmo di Nogara and Fondo Paviani) show higher δ 15 N and δ 13 C values compared with the sites from the center and the south (Fig. 3). In addition to the environmental and ecological differences which affect the values from the bottom of the trophic chain, the 15 N enrichment in the northern animals can be interpreted as them grazing close to lands with manured crops or being fed manured plants. The carbon data fit within the typical range of values from a C 3 environment, with the exceptions of Fondo Paviani, that show enriched δ 13 C values suggesting a C 4 plants intake, being likely fed food waste 25 .
Given the variability in the animal results, the human diets are compared using the offsets between the humans and their associated fauna. The Δ 15 N h-f span from 0.6‰ at Gr. dell' Antenato to 2.9‰ at Gr. Misa, below what is normally estimated between two trophic levels. As for the northern Italy, Olmo di Nogara and Bovolone, the Δ 15 N h-f are 2.0‰ and 2.4‰ respectively, indicating that the animal protein intake was not very important in this area. The lowest offsets are found in the center and the south of the peninsula, suggesting a limited consumption of animal foodstuffs and supporting a diet mainly based on cereals and legumes. The consumption of legumes seems to have been considerable in central Italy during the Bronze Age and the variety of species is greater than in the Neolithic 16 , as attested by the remnants of a soup of barley, oat, einkorn/emmer wheat and broad beans found at Gorgo del Ciliegio 96 . Therefore, if during the EBA the site of Scoglietto does not show evidence for legume consumption neither from an archaeobotanical nor an isotopic perspective, the δ 15 N values from Felcetone, Gr. Vittorio Vecchi and Gr. Misa confirm the consumption of pulses from the MBA. Additional data from La Pastena cave support the importance of pulses 86 . Based on these results, the MBA represents an www.nature.com/scientificreports/ important moment of change in terms of the adoption of new foodstuffs for central Italy. For the south of the peninsula, archaeobotanical research supports an increase in the pulse varieties, although broad beans still continue to represent the main taxon 7 . Consequently, according to the nitrogen data for the MBA sites of the entire peninsula, animal protein was not highly consumed during this period, revealing an economy more oriented towards vegetal products rather than animal products. Regarding the δ 13 C data, interesting patterns appear. The δ 13 C human values are widespread, from − 20.7 to − 10.6‰. A sligh variability has already been identified at the end of the EBA, but at the MBA this pheonomenon is accentatued. Three main groups with specific dietary patterns can be identified. The first includes sites from central and southern Italy: Trinitapoli, Lavello, Gr. dell' Antenato, Gr. della Monaca, Toppo Daguzzo, Gr. Regina Margherita and Gr. Vittorio Vecchi. Here, the Δ 13 C h-f are between 0.1 and 1.0‰, the characteristic enrichment between two trophic levels. Furthermore, the δ 13 C values of these humans overlap and fall within a limited range, from − 20.7 to − 18.7‰, suggesting C 3 plant consumption. The second group is composed by Gr. Misa and Felcetone. These two communities have common characteristics. First, both sites show wide Δ h-f 13 C offsets, (GM = 2.7‰ and FEL = 1.8‰) and, second, they show a wide intragroup δ 13  13 C enrichment implies that these crops were mostly ingested directly. According to these data, different trends are evident for the MBA. Some of them are present in the whole peninsula and others are only regional. A general low animal protein intake is detected in all the human groups of this period. In fact, most of the sites do not reach the Δ 15 N h-f offset of 2‰. The greatest inter-and intragroup dietary differences are recorded in the north. Here, diet is mainly based on C 4 plants (Olmo di Nogara and Bovolone), with evidence for millet consumption also seen among the animals (Fondo Paviani). In central Italy, C 4 plants started to play an important role in the diet from this period. Nevertheless, C 3 plants remained the staple food (Felcetone, Gr. Misa, Gr. Regina Margherita and Gr. Vittorio Vecchi) with only some individuals apparently consuming C 4 plants (Felcetone and Gr. Misa). Furthermore, it seems that pulses were an important part of the diet in this area (Gr. Vittorio Vecchi, Felcetone), as recently supported by other studies as well 86 In the south, there is a lower intra-and intergroup dietary variability and C 4 plants were not routinely consumed (Toppo Daguzzo, Lavello, Trinitapoli, Gr. Della Monaca and Gr. dell' Antenato). It is, therefore, probable that millets started to be routinely consumed from the north to the south of the peninsula, with a likely gradual replacement of the more common wheat and barley. The animal nitrogen and carbon values are widespread and significant differences occur among the groups (Supplementary Table A .5). This wide variability is particularly evident for carbon values because the animals from P. di Zambrone, at the south of the Peninsula, are enriched in 13 C compared to the ones from the north. Even though differences linked to the diverse environmental conditions could not be excluded because of the different latitudes, the results are more likely related to different diets. The specimens from the north had a diet mainly based on C 3 plants, whereas the ones from the south, at least some of them, grazed upon seaweeds or, more likely, had a mixed C 3 -C 4 plants consumption, in accordance with the archaeobotanical remains of P. di Zambrone 97 (Fig. 4).
According to the human and animal offsets, different human dietary habits are evident. If the Δ 15 N h-f and Δ 13 C h-f at P. di Zambrone are not remarkable, suggesting that both humans and animals had a C 3 -C 4 mixed terrestrial diet, the ones from Ostiglia and Buco del Diavolo are considerable, 8.0‰ and 2.1‰ respectively, indicating a human inter-and intragroup food variability and the significant consumption of 13 C enriched resources. As mentioned, millets started to be consumed from the MBA and these results demonstrate that this trend continues later. Furthermore, humans mainly consumed C 4 plants directly, since animals do not show any C 4 signal. At Ostiglia and Buco del Diavolo, the Δ 15 N h-f are 4.4‰ and 4.0‰ respectively, suggesting that animal protein and/or aquatic resources had an important role in the diet. Taking into account the geographical area of the two sites, Buco del Diavolo in Liguria, over 1000 m. a.s.l., and Ostiglia, in the Po Basin, in the center of one of the most economically developed areas during the Bronze Age, it is not surprising that such a wide variety of resources was consumed. In addition, studies on animal management during the FBA in the Po Basin show that even if the subsistence economy was based on agricultural activities, pigs were slaughtered before reaching adulthood to exploit the higher quality of meat and cattle and caprines were reared until adulthood to exploit their secondary resources 42,98 . These results, which are consistent with animal protein intake, indicate that specific attention was paid to animals depending on their resources and their different purposes.
The great diversity in the dietary practices for the RBA-FBA compared to the previous phases is present at the intra-group level as well, particularly at Buco del Diavolo, as previously described. This is the first time that such different dietary strategies are recorded between individuals from the same community in the Italian Peninsula. The consumption of C 4 plants represents the first major change in terms of new crops since the introduction of agriculture, and it has also been identified as an example of "food globalization" 21 . In order to trace the origins of this new practice, botanical research together with isotopic data from all of Italy have been evaluated. Some of the archaeobotanical evidence supports the spread of millets starting in the Neolithic and throughout the Bronze Age. First, there is a significant increase in the number of sites where millet can be found (Fig. 5 and Supplementary Information 2, Table B). Second, the distribution area is wider, including sites in southern Italy. Third, the findings are varied, representing both seeds and chaff. The greatest amount of millets has been found in the Po Basin, highlighting how crucial this zone was for the spread of millet throughout the peninsula (Fig. 5 and Supplementary Fig. A.4).

Insight into the changes in Bronze
Isotopic studies in Italy show no evidence of C 4 plant consumption for the Neolithic 65,71,72,98-105 , even if doubtful consumption seems to have been recorded in some individuals at Mora Cavorso 106 . Neither were millets commonly consumed during the Eneolithic [106][107][108][109][110] . Even if an enrichment in 13 C could be induced by several causes, i.e. marine foodstuffs, from this study it is evident that increasing higher δ 13 C values are mainly due to C 4 plants. The first evidence for C 4 plants intake is detected in the EBA and EBA-MBA in the northeast of the peninsula-Dossetto and Sedegliano. All other evidence dates to the MBA in northern Italy-Olmo di Nogara and Bovolone and to a lesser extent in the central regions-Gr. Misa and Felcetone. The C 4 signal is well established later on in the Bronze Age, the RBA-FBA, mainly in the north-Ostiglia, Buco del Diavolo, with some evidence for the south at P. di Zambrone (Figs. 6 and 7).
When combining isotopic and archaeobotanical data, a more detailed scenario emerges for the identification of the "Italian millet road". Where botanical studies infer the presence/absence of C 4 plant species, stable isotopes analyses quantify the direct or indirect intake of these plants. In fact, archaeobotanical remains reveal the spread of millets from northern Italy since the Neolithic (Fig. 5), but the isotopic data indicates humans only started to regularly consume millets from the EBA-MBA transition, beginning in the northeast, then the center and finally the south of the peninsula (Fig. 7). Radiocarbon dates performed on charred broomcorn millet grains confirm this trend. Filipović et al. 21 estimate the start of millet cultivation in the Po Basin between 1570 and 1410 cal. BC (95.4% probability) at the beginning of the full development of the Terramare culture 111 . Furthermore, biomolecular analysis performed on on-site archaeological sediments (i.e. Fondo Paviani) confirms the presence of broomcorn millet by the biomarker miliacin 112 . Millets were probably consumed in previous phases, like the dental calculus from Gr. Scoglietto suggests 94 , but in such a small quantity that it cannot be isotopically detected.
In southern Europe, the first evidence for millet consumption comes from Greece at Perachora 113 in the Early Helladic (3100-2100/2050 BC) and at Rymnio and Spathes 114 , Pineiada, Aghia Triada and Almyri [114][115][116][117] dated to the Late Helladic (1700/1675-1075/1050 BC). Croatia, Spain and France, as well as countries beyond the Alps, like Switzerland, Germany and Austria, show sporadic C 4 signals, but only from the Late Bronze Age onwards. Conversely, during the IA, results are more homogeneous exhibiting the typical values of millet consumers 76,117-127 . www.nature.com/scientificreports/ It seems that the Caucasus area represents a key zone for the spread of millets into Europe because millet intake is recorded from the first half of the 2nd millennium BC 128,129 . The identification of an 'Isotopic Millet Road' from China to Central Europe and the Mediterranean reveals that C 4 dietary signatures reflect early links, in the form of migration and/or resource transfer, between the Bronze Age inhabitants of China and Europe 130 . These results show that the westward expansion of millet as a foodstuff into Europe was a rather fast and global phenomenon instead of a gradual one. This picture is confirmed by Filipović et al. 21 , whose research indicates the earliest occurrence of millet at the middle of the 2nd millennium BC, supporting its spread "at only slightly different times in different parts of Europe". Their Bayesian model shows that it was cultivated in southeastern Europe (Ukraine) at the earliest during the sixteenth century BC, promptly spreading during the fifteenthfourteenth centuries BC to most of southeast and Central Europe and only in the late 13th/early twelfth centuries BC into northwest Europe. Consequently, the radiocarbon-dated caryopses indicate that millet did not arrive in Europe during the Neolithic and that most of the Neolithic attributions should therefore be doubted. Furthermore, considering regional variability, the first millet consumption in Europe, including Italy, and the earliest radiocarbon dates on broomcorn millets seems to overlap. This evidence suggests that the adoption of millet as part of a human diet was heterogeneous and was contingent on the area and the site.
Why are new crops like millets introduced? An important demographic growth associated to an increasing socio-political and economic connectivity between the Mediterranean area and continental Europe may provide the answers. This connectivity has been crucial not only for the exchange of ideas, lifestyle and technologies but also for objects, products (crops and animals) and resource transfer (e.g., raw materials, prestigious goods), as demonstrated by archaeological evidence and confirmed by bioarchaeological and genetic data [130][131][132][133] . Contacts between the Carpathian basin and northern Italy, through Austria and Slovenia, have been well identified, particularly from the Bronze Age 134 , and millet was probably part of this "package exchange". Millets were likely  www.nature.com/scientificreports/ introduced to diversify the agricultural production and to ensure harvests when annual winter wheat and barley crops were not sufficient.
Pivotal agricultural system changes have been recorded since the Eneolithic: these mainly consisted in the transition from a self-sufficient horticultural subsistence strategy to a more extensive agricultural system, where intensive production was managed by a more complex society and when a gradual demographic growth is recorded. The ever increasing areas dedicated to agricultural and pastoral activities caused heavy deforestation with the over-exploitation of land and caused the soil to dry up. This aridity led to the introduction of innovative strategies, including a network of irrigation ditches as well as the cultivation of less demanding plants, such as millets 14,135 .
In addition, episodes of dryness are recorded in the EBA, as different proxies in the Italian sub-alpine lakes show 84,136 , they are also observed as a prominent warm spell in the residual ∆ 14 C curve. These short events could have affected variations in crop cultivation. Therefore, the increasing aridity caused by climatic fluctuations associated to heavy deforestation and the over-exploitation of soils 12,14,137 , supports the introduction of new, more tolerant species, less sensitive to stressful conditions. However, some questions arise: why was millet mainly consumed only in the north of Italy? Why was millet differently consumed within groups (e.g., Buco del Diavolo)? According to the evidence from other European sites, variable intake could be related to social, cultural and geographical factors 118,[120][121][122][123]127 . Indeed, millet consumption is attributed to lower social status individuals in Croatia and Germany 76,123 . Nevertheless, the C 4 signal, when detected in the Italian sites, is usually recorded for all the community's individuals, like at Olmo di Nogara, Bovolone and Ostiglia, even if this last site is just represented by few individuals. It is, therefore, likely that millet was easily accessible in Italy.
The second phenomenon concerns the growing importance of legumes both in diversifying the human diet and for improving the fertility of the soil through crop rotation 9 . Pulse cultivation was mainly based on broad bean all across Italy and, depending on the site, significant quantities of pea, grass pea, red pea, lentil, common vetch and ervil have been found 6 . Indeed, in addition to a greater abundance of legume seeds, there is also a wider variety of species, especially compared to the Neolithic 138 . If this picture is quite clear for the north, it is less evident for the center and the south of Italy. Despite the increasing number of taxa, the presence of legumes is often controversial. Sometimes it is not possible to ascertain whether these plants were weeds or were cultivated for their physiological characteristics 6,7 . Isotopic analyses contribute to discriminating between legumes as part of the diet or not and determining whether they were cultivated or not. Indeed, legume consumption can be detected when associated with a low animal protein intake. This is the case of Gr. Vittorio Vecchi. Furthermore, the archaeobotanical data from this site show that bean seeds represent more than 50% of the total remains 16,139 , revealing the significance of legumes for this community. Consequently, these new data, associated with other coeval sites of the region and additional unpublished data 86,110 , provide evidence for the increasing importance of legume consumption during the Bronze Age for central Italy. This site clearly showcases how multidisciplinary studies that combine isotopic data and archaeobotanical research are essential for the reconstruction of subsistence practices and for defining the significance of these plants in human diet.
Finally, the third evidence is a rise in animal protein consumption. Indeed, a gradual enrichment in 15 N in the individuals of the northeast, northwest and in the south throughout the Bronze Age is recorded (Fig. 6). The higher nitrogen values could be attributed to different causes, like the consumption aquatic resources as  Cereal cultivation significantly declines, while the uncultivated areas spread 142 . It is therefore possible that several years of dryness, in conjunction with economic and social factors, contributed to precipitating a situation that was already beyond the thresholds of sustainability, pushing the population to invest in new resources, like in a pastoral economy for meat and secondary products. Even though the species exploited as well as the animal size varied according to geographical area 143 , research into livestock farming in Liguria, for instance, supports an increased interest in the pastoral economy. During the Neolithic, during times of important demographic growth, pastoral practices, which included the use of caves as stables and suckering to provide foliar fodder, were introduced 144,73 . From the end of the Neolithic, transhumant pastoralism spread and contributed to the thinning out of coniferous forests in the mountains to obtain, through the practice of controlled fires, new highland pastures 17 . During the Eneolithic and Bronze Age, when the climate deteriorated, the practice of transhumance towards highland pastures was consolidated 145 . Figure 7. The "Italian millet road". Map of Bronze Age Italian sites for which isotopic analyses have been carried out. The color intensities indicate differential consumption of C 4 plants according to chronological period (The map is elaborated using "Natural Earth. Free vector and raster map data @naturalearthdata.com" available at https:// www. natur alear thdata. com/ downl oads/ 10m-raster-data).

Conclusions
This research presents the first global overview of human and animal isotopic data used to reconstruct dietary patterns for Bronze Age Italy. As some areas are still isotopically unexplored, the present scenario may change with new studies. However, based on the present data, some trends have been highlighted. Results suggest that the MBA was a period of major change and that the north of Italy, particularly the northeast, seemed to have been at the forefront of this dynamic, where new trends were introduced that spread into other areas of the peninsula. Changes in dietary patterns are clearly detected, resulting in either global or more regional patterns. Indeed, we highlighted (I) an increased consumption of broomcorn and foxtail millet, mainly for humans from the EBA/MBA transition, (II) a greater legume consumption, particularly for the populations of the center and south of Italy from the MBA onward and (III) a likely increase in meat and/or dairy product intake at the end of the Bronze Age. Several factors could have been responsible for these changes, including the climatic fluctuations that resulted in more arid soils, alongside the strong impact of human activities. Indeed, anthropic activities like deforestation, the over-exploitation of soils for agriculture and livestock breeding and the increase of metallurgic production could have reduced soil fertility. Consequently, the need for a more diverse diet arose, leading to the adoption of more tolerant crops and further resources to exploit. In addition, the increase of trade and the related economical activities with other Mediterranean and continental European areas points to exchanges at different levels, not only in terms of prestigious goods, i.e. ivory, amber and silk, but also in terms of local agricultural practices, ways of life, traditions and, consequently, socio-economic systems.
This study is a first step towards a more in-depth understanding of the Italian dietary patterns during the Bronze Age, which is still a largely unexplored period in terms of biomolecular investigations, and it represents also a new piece of the gigsaw of the global understanding of Bronze Age societal developments. Further isotopic data on botanical, animal and human remains are necessary to improve the resolution of our analyses and to gain a more fine-grained depiction of subsistence strategies during a time of rapid socio-economic change.

Methods
Carbon and nitrogen isotope analyses on human and animal bone collagen. Protocols proposed by Longin et al. 146 and modified by Bocherens 147 were followed for bone collagen extraction for Pertuso, Buco del Diavolo, Ostiglia La Vallona, Gr. Vittorio Vecchi, Trinitapoli and Castiglione. The collagen extraction was performed at the LAMPEA Biochemistry Unit (Aix-en-Provence, France) and the isotopic ratios were measured at Iso-Analytical Ltd (Europa Scientific 20-20, Crewe, UK). The isotope ratios are reported as delta that has been defined according to IUPAC (International Union of Pure and Applied Chemistry): where Rs and Rst are the isotope ratios 15 N/ 14 N and 13 C/ 12 C of the isotopic abundances of 15 N, 14 N, 13 C, and 12 C, and ‰ = 10 -3 . The analytical precision of both samples and standards is 0.1‰ for both δ 15 N and δ 13 C.
Analytical method. 0.5 g of powdered bone was soaked in HCl (1 M, 20') for demineralization and in NaOH (0.125 M, 18 h) to eliminate contaminants and finally dissolved in HCl (pH = 2, 17 h, 100 °C). The residue was then frozen (> 5 h, − 60°) and freeze dried for 48 h. Isotopic ratios and elemental contents were provided by Elemental Analyzer coupled to Isotope Ratio Mass Spectrometry at Iso-Analytical Ltd (Europa Scientific 20-20, Crewe, UK).
Isotopic accuracy was monitored through routine analyses calibrated against international standards: bovine liver (IA-R042), a mixture of ammonium sulfate (IA-R045) and beet sugar (IA-R005), and a mixture of sugar cane (IA-R006) and ammonium sulfate (IA-R046). The analytical precision of both samples and standards is 0.1‰ for both C and N. Several preservation criteria have been considered to check for collagen integrity: collagen yields greater than 1%, %C ≥ 30% and %N ≥ 10%, and C/N ratios ranging between 2.9 and 3.6 146-150 . Statistical analysis. Statistical analysis used R 3.6.1 151 and the coin package v. 1.3-1 152 . Considering the small sample size, the non-parametric tests of Kruskal-Wallis (Monte Carlo approximation, 10,000 replicates) and exact Wilcoxon Mann-Whitney are used to assess for significant differences in δ 13 C and δ 15 N. In the case of multiple tests, in order to identify as many significant comparisons as possible while still maintaining a low false positive rate, the False Discovery Rate (FDR) is applied on the results obtained by the Wilcoxon Mann-Whitney tests 153 . Only corrected p-values are presented. A 0.05 probability (p < 0.05) is considered significant.

Data availability
All data needed to evaluate the conclusions in the paper are present in the paper and in the Supplementary Information 1 and 2.